Design, synthesis and biological evaluation of (E)-3,4-dihydroxystyryl 4-acylaminophenethyl sulfone, sulfoxide derivatives as dual inhibitors of HIV-1 CCR5 and integrase

Article abstract of DOI:10.1016/j.bmc.2016.12.035

Aiming at the limited effectiveness of current clinical therapeutic effect of AIDS, novel series of compounds bearing (E)-3,4-dihydroxystyryl sulfone (or sulfoxide) and anilide fragments were designed and synthesized as dual inhibitors of HIV-1 CCR5/IN. T

Full text of DOI:10.1016/j.bmc.2016.12.035

Accepted Manuscript
Design, Synthesis and Biological Evaluation of (E)-3,4-Dihydroxystyryl 4-
Acylaminophenethyl Sulfone, Sulfoxide Derivatives as Dual Inhibitors of
HIV-1 CCR5 and Integrase
Yixing Sun, Weisi Xu, Ningning Fan, Xuefeng Sun, Xianling Ning, Liying Ma,
Junyi Liu, Xiaowei Wang
PII:
S0968-0896(16)31442-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.12.035
BMC 13462
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 August 2016
26 November 2016
16 December 2016
Please cite this article as: Sun, Y., Xu, W., Fan, N., Sun, X., Ning, X., Ma, L., Liu, J., Wang, X., Design, Synthesis
and Biological Evaluation of (E)-3,4-Dihydroxystyryl 4-Acylaminophenethyl Sulfone, Sulfoxide Derivatives as
Dual Inhibitors of HIV-1 CCR5 and Integrase, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.12.035
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Design, Synthesis and Biological Evaluation of
(E)-3,4-Dihydroxystyryl 4-Acylaminophenethyl
Sulfone, Sulfoxide Derivatives as Dual Inhibitors of
HIV-1 CCR5 and Integrase
Yixing Suna,‡, Weisi Xub,‡, Ningning Fana, Xuefeng Suna, Xianling Ninga,
Liying Mab,*, Junyi Liua,c,* and Xiaowei Wanga,*
a Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China.
b
National Center for AIDS/STD Control and Prevention (NCAIDS), Chinese Center for Disease Control and
Prevention, Beijing 102206, China
c
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
* Corresponding authors. Tel.: +86 10 82801706 (J.L.); tel.: +86 10 82805203 (X.W.). E-mail addresses:
jyliu@bjmu.edu.cn (J. Liu), xiaoweiwang@bjmu.edu.cn (X. Wang), mal@chinaaids.cn (L. Ma).
‡ These Authors contribute equally.
ABSTRACT: Aiming at the limited effectiveness of current clinical therapeutic effect of AIDS, novel series of
compounds bearing (E)-3,4-dihydroxystyryl sulfone (or sulfoxide) and anilide fragments were designed and
synthesized as dual inhibitors of HIV-1 CCR5/IN. The biological results indicated that several target compounds
showed inhibitory activity against HIV-1 Bal (R5) infection in TZM-bl cells. Besides targeting the chemokine
receptor on the host cell surface, they also displayed binding affinities with HIV-1 integrase using the surface
plasmon resonance (SPR) binding assays. Molecular docking studies have inferred the possible binding mode of
target compounds against integrase. These data demonstrate that the structure of (E)-3,4-dihydroxystyryl sulfone and
sulfoxide derivatives have the potential to derive potent dual inhibitors of HIV-1 Integrase and CCR5.
KEYWORDS: HIV dual inhibitors; CCR5 receptor; HIV integrase; Sulfones; Sulfoxides.
1. Introduction
In early stages of the development of highly active anti-retroviral therapy (HAART, a.k.a. “drug
cocktails”), the treatments of HIV were limited to the combination therapies of reverse transcriptase
(RT) inhibitors and protease inhibitors. With the application of integrase (IN) inhibitors, fusion
inhibitors and co-receptor inhibitors, not only the selection scope of HAART has been widened, but
the treatment outcome has also been improved.1 Nevertheless, the poor patient compliance, the risk
of drug interactions, and the emergence of resistant HIV strains brought by HAART have promoted
to discovering new strategy against HIV, which include designing multi-targeted inhibitors.2,3
In order to block the HIV replication cycle at different stages, we planned to develop dual
inhibitors of HIV-1 CCR5 and integrase. During the integration process in HIV life cycle,
chemokine receptors CCR5, along with CXCR4, are required for virus’ membrane fusion with host
cell.4 In particular, the CCR5 has a main mediating role in the initial infection of HIV.5 Also, with
the action of integrase, cDNA strands assemble the host genomic DNA into a double-stranded DNA

to complete replication.6 Additionally, the integrase serves as a feasible intracellular target since it
hasn’t been found in mammalian cells.7 Accordingly, targeting both the integrase and CCR5 may
provide low toxicity as well as great efficiency and timeliness.
Recent studies in the crystal structure of the CCR5-maraviroc complex have demonstrated the
multiple forces between the receptor and the ligand.8 These include a salt bridge interaction
between the protonated tropane nitrogen and E283, several hydrogen bond forces including amide
nitrogen to Y251, as well as the hydrophobic contacts between the phenyl group with the aromatic
amino acid residues. In the meantime, previous crystallography studies have demonstrated the
binding mode of raltegravir (RAL) with the Prototype Foamy Virus IN. The di-carbonyl moiety of
diketo acids (DKAs) changed the conformation of IN by coordinating to the divalent ions in the
active domain of the protein.9,10
Caffeic acid phenethyl ester (CAPE) has also been reported to inhibit the process of
integration.11,12 As shown in SAR studies, diphenol and carbonyl units are necessary to maintain the
IN inhibitory activity.13,14 Due to the susceptibility of carboxylic ester to hydrolase, bioisosteric
replacement took place and sulfone (or sulfoxide) group was brought in to form the structure.
Sulfone and sulfoxide groups are not only more stable than ester groups, but also more soluble in
water. Moreover, Smithkline Beecham Corp. disclosed various benzanilides to acquire CCR5
inhibitory properties through generating corresponding hydrogen bonds and hydrophobic
interactions.15,16 With these evidences in mind, we highly integrated the structures of sulfone and
sulfoxide derivatives of CAPE and aroyl aniline moiety which could acquire CCR5 inhibitory
properties through generating corresponding hydrogen bonds and hydrophobic interactions, and
formed new series of target compounds (Fig. 1).
Figure 1. Design of dual inhibitors based on derivatives of CAPE and potent CCR5 antagonists.
Herein, we proposed to synthesize the target compounds to probe their antivirus activity against
R5 HIV-1 strains and IN binding affinity. These results might provide useful information for
developing novel dual inhibitors of HIV-1 IN and CCR5.
2. Results and discussion

2.1. Chemistry
For the preparation of the sulfone and sulfoxide target compounds, the synthetic routes (shown in
Scheme 1) were explored respectively. The synthesis of 4, 5, and 6 were based on our previous
report.17 Commercially available 4-nitrophenethyl bromide was reacted with mercaptoacetic acid to
obtain compound 4, which was then oxidized with H2O2 in different solvents to afford 5 and 6
respectively.
In the presence of β-aminopropionic acid as catalyst, the sulfonyl intermediate 7 was obtained
with high yield through Knoevenagel condensation of compound 5 with acetyl protected
3,4-dihydroxybenzaldehyde. However, pyrrolidine and HOAc was used as catalysts to give the
sulfinyl intermediate 8. The change of the Knoevenagel reaction condition is due to the decreased
reactivity of the methylene structure.
Following the Knoevenagel condensation reaction, the nitro group of 7 was selectively reduced
with palladium-catalyzed hydrogenation to afford the aromatic amine 9. Due to the decrease of
Pd/C catalytic activity that caused by the existence of the sulfoxide group, sodium hydrosulfite
was used as reductant to obtain the intermediate 10. Reaction of 9 and 10 with various acid
chlorides gave the corresponding compounds 11a-i and 12a-f. Final deprotections of the acetyl and
MOM groups gave the target compounds 13a-i and 14a-f, respectively.

Scheme 1. Synthetic routes of target compounds.
Reagents & conditions: (I) NaOH, MeOH, r.t., 2h, yields 80-93%; (II) 30%H2O2, HOAc, r.t., yields 74-97%; (III)
30%H2O2, MeOH, r.t., yields 83-95%; (IV) β-aminopropionic acid, THF, 4Å MS, reflux, 12h, yield 67-80%; (V)
pyrrolidine, HOAc, THF, 4Å MS, reflux, 12h, yield 51-71%; (VI) H2, Pd/C, CH2Cl2, r.t., 3h, yield 64-82%; (VII)
Na2S2O4, EtOH, H2O, reflux, 12h, yield 25-45%; (VIII) RCOCl, Et3N, CH2Cl2, ice bath, 2h, 16-75%; (IX) K2CO3,
MeOH, r.t., 3h, yield 56-65%; (XI,XII) RCOCl, Et3N, CH2Cl2, ice bath, 2h; HCl, MeOH, reflux, 3h, total yield
10-23%.
2.2. Biological results
2.2.1. Determination of the cytotoxicity and antivirus activity against R5 HIV-1 strains
All the target compounds were sequentially tested for their cytotoxicity and their antivirus
activity against R5 HIV-1 strains in TZM-bl cell line. For the in vitro entry inhibition assays, HIV-1
Bal (R5) virus strain was used to determine the type of chemokine receptors that mediate virus’
entry.
The TZM-bl cell line was selected to complete the cell based assays. Reporter genes such as
firefly luciferase gene, which is promoted by HIV long terminal repeat sequence (LTR), and E. coli

β-galactosidase gene are carried and receptors including CCR5 were expressed simultaneously by
this cell line. The intracellular viral load can therefore be tested by a fluorescence detector after the
virus’ entrance to the cell. Due to the specific mediation of CCR5 during the virus’ entrance, HIV-1
Bal (R5) virus strain was used for measuring the CCR5 inhibitory activity of the target compounds.
All target compounds were tested for their cytotoxicity before testing for cell based anti-HIV
activity. As shown in Table 1, the CC50 values of most active compounds are above 100μM.
Meanwhile, the sulfone and sulfoxide derivatives at 50μM concentration displayed antiviral activity
against R5 HIV-1 strains at different levels. Five compounds, including four sulfoxide derivative,
showed at least 50% inhibition of HIV-1 replication in TZM-bl cells. The preliminary biological
results suggested that sulfoxides showed better activity than sulfones. Besides, the difference of R
substitution may affect the CCR5 inhibitory activity. Herein, 3,5-difluorophenyl group, cyclohexyl
group, 4-nitrophenyl group, 1-adamantaneformyl group, and 4-fluorophenyl group are the more
favored substituent groups.
Table 1. Inhibition of HIV-1 entry through CCR5 co-receptor as analyzed in TZM-bl cells using HIV-1 Bal (R5)
virus
Compound
11a
R
R’ Series
% Inhibitiona CC50b (μM) Compound
R
R’ Series
% Inhibitiona CC50b (μM)
Ac Sulfone
Ac Sulfone
Ac Sulfone
Ac Sulfone
Ac Sulfone
Ac Sulfone
Ac Sulfone
Ac Sulfone
Ac Sulfone
<20
<20
26.2
<20
<20
23.7
<20
52.2
23.0
108
858
162
225
169
182
177
160
161
157
ND
127
13a
13b
13c
13d
13e
13f
13g
13h
13i
H
H
H
H
H
H
H
H
H
H
H
H
Sulfone <20
Sulfone <20
238
11b
>1000
>1000
414
11c
Sulfone <20
Sulfone 33.0
Sulfone 45.9
Sulfone 21.6
Sulfone <20
Sulfone 23.4
Sulfone <20
Sulfoxide 41.8
Sulfoxide 52.7
Sulfoxide 64.7
11d
11e
638
11f
786
11g
168
11h
483
11i
569
14a
H
Sulfoxide 68.4
Sulfoxide 34.1
Sulfoxide 64.6
14d
14e
14f
ND
14b
14c
H
ND
H
ND
Maraviroc --
-- --
IC50=0.2nM >1000
ND = Not determined.
a % Decrease in fluorescent intensity in TZM-bl cells; data represent mean values for two wells.
b CC50 = concentration of compound required for 50% cytotoxicity against uninfected cells.
2.2.2. Assays for measuring the in vitro binding affinity of target compounds against HIV-1 IN

Having determined the antivirus activity against R5 HIV-1 strains, we carried out assays for the
second target. In order to investigate the binding affinity of target compounds against HIV-1 IN, we
used SPR based Biacore T200 biosensor for evaluation. The commercially available recombinant
IN protein was immobilized on a CM5 chip and the binding response was recorded continuously in
the response unit (RU) and graphically presented as a function of time. Preliminary screening of the
24 compounds showed that compound 11a, 13a, 13e, and 14a have better binding results (shown in
Fig. 2). Then the dose-dependent relations was tested with various concentrations for the four
compounds (200μM, 100μM, 50μM, 25μM, 12.5μM, etc.). To determine the equilibrium
dissociation constant (KD), the 1:1 binding fit model (kinetics) was used. According to the affinity
estimates, the KD value of compound 14a was 141.8μM. As shown in Table 2, these SPR results
determined that the (E)-3,4-dihydroxystyryl sulfone and sulfoxide derivatives have binding affinity
against HIV-1 integrase.
Table 2. KD values of the 4 representative compounds
Compound
11a
13a
13e
14a
KD(μM)
207
160
115
142
Figure 2. HIV-1 IN binding affinity using SPR biosensor technology. Representative sensorgram of 11a(A), 13a(B),
13e(C), 14a(D) were obtained.
It is noteworthy that compound 14a demonstrate potency activity against R5 HIV-1 strains with
relatively low toxicity to TZM-bl cells and moderate binding affinity with IN (KD = 141.8μM).
Therefore, the preliminary results suggested that target compound 14a could be potent dual inhibitor
of HIV-1 CCR5/IN.

2.3. Molecular docking
To assess the dual inhibitory properties of the target compounds, computational docking studies
were carried out to explore the binding mode of the target compounds with active sites of CCR5 and
prototype foamy virus (PFV) IN respectively.
Firstly, in docking studies with CCR5 (Fig. 3) (PBD: 4MBS), the results revealed that H bonds
were observed between the phenolic oxygen and Tyr37, as well as amide nitrogen and Glu283. The
bonding interactions have significant similarities with results from CCR5-maraviroc complex
studies which are supposed to assist the molecule’s positioning in the binding pocket,7 and are
recognized as the key points of exhibiting CCR5 inhibitory activity. The phenyl rings of inhibitors
imparted π-π stacking with the CCR5 active site residues (Trp86, Tyr108, Phe109, Phe112, etc.).
Additionally, hydrophobic interactions were developed by the positioning of aromatic rings into the
hydrophobic pockets comprised by aromatic residues Tyr108, Phe109, Phe112, Trp248, and Tyr251.
Figure 3. Molecular docking result of compound 14a into HIV CCR5 (PBD: 4MBS). Hydrogen bonds were signed
in yellow dotted line.
To further explore the potential of IN inhibitory activity of these compounds, docking works were
performed using the PFV IN core domain (Fig. 4). Compound 13e exhibited H bonding between one
of the phenolic oxygen and the side chain of Glu221 residue, as well as coordination between the
catechol oxygens and metal ion Mg2+ that results in the chelation. Meanwhile, in docking results of
compound 14a, Mg2+ metal ions coordinate with sulfinyl oxygens while H bonds are generated
between acyl oxygen and Tyr212.

Figure 4. Molecular docking of compound 13e (left), and 14a (right) into PFV IN (PBD: 3OYA). The balls colored
in green represents Mg2+ ions.
Therefore, the binding modes of the target compounds against HIV CCR5 and PFV IN were
preliminarily revealed, laying a foundation for further development of these structures.
3. Conclusion
To sum up, series of (E)-3,4-dihydroxystyryl 4-acylaminophenethyl sulfones and sulfoxides were
synthesized and tested for their in vitro HIV-1 cellular CCR5 inhibitory activity (close to 68.37% at
50μM) and binding affinity against HIV-1 integrase (KD at around 10-4 mole) . The two independent
mechanism enable the target compounds to block the entry and integration stages respectively, and it
would possibly reduce the generation of drug resistant viruses. Taken the molecular docking results
together, our study identified that the (E)-3,4-dihydroxystyryl sulfone and sulfoxide derivatives
could derive novel compounds that display dual inhibition activities for HIV-1 virus.
4. Experimental procedures
4.1. Chemistry
4.1.1. General chemistry methods
The structural characterizations of compounds were performed by NMR, which were recorded on
Bruker AVANCE III-400 spectrometer (400MHz) with TMS (tetramethylsilane, Me4Si) as an
internal standard. Chemical shifts were reported in δ (ppm) while coupling constants were measured
in Hz. CDCl3 and DMSO-d6 were used as the solvents for NMR experiments. The purity of target
compounds was determined by HPLC (Agilent 1260 infinity HPLC system). All the assayed
compounds showed >95% purity by HPLC. All reactions were monitored by TLC, which was
performed on precoated silica gel GF254 plates. The detection of TLC was done by UV light
irradiation (UV lamp, model UV-IIB). Column chromatography was performed by silica gel H
(200-300 or 500 mesh). Melting points were measured on an X-4 apparatus without correction. THF
were purified and dried by standard methods. All other organic solutions were dried over anhydrous
sodium sulfate. All other reagents were purchased as reagent grade from commercial sources and
without further purification unless otherwise stated.
4.1.2. Synthesis of (E)-3,4-diacetoxystyryl 4-nitrophenethyl sulfone (7)
To a solution of 2-(4-nitrophenethylsulfonyl) acetic acid (5, 3mmol) in THF (15mL),
3,4-diacetoxybenzaldehyde (2mmol) and β-aminopropionic acid (1mmol) were added and the
resulting solution was heated to reflux until absence of 3,4-diacetoxybenzaldehyde (checked by
TLC). The solvent was evaporated under vacuum and the mixture was diluted with H2O and
extracted with ethyl acetate. The combined organic fractions were washed with brine, dried with
sodium sulfate, and concentrated in vacuo. Purification of the crude product by column
chromatography (PE/EA) afforded the title compound 7. Yield: 80%; Yellow solid; mp 86–87 ºC; 1H

NMR (400MHz, CDCl3) δ 8.17 (d, 2H, J = 8.5 Hz), 7.55 (d, 1H, J = 15.4 Hz), 7.27-7.41 (m, 5H),
6.72 (d, 1H, J = 15.4 Hz), 3.38 (m, 2H), 3.25 (m, 2H), 2.32 (s, 6H); 13C NMR (100MHz, DMSO-d6)
δ 168.9, 168.7, 145.4, 145.1, 143.9, 143.2, 133.7, 132.5, 130.1, 130.1, 127.2, 124.3, 124.1, 123.9,
123.9, 122.8, 56.0, 28.3, 20.5, 20.5; MS (ESI) m/z 456.05 [M+Na]+.
4.1.3. Synthesis of (E)-1,2-bis(methoxymethoxy)-4-(2-((4-nitrophenethyl)sulfinyl)vinyl)
benzene (8)
To a solution of 2-(4-nitrophenethylsulfinyl) acetic acid (6, 3mmol) in THF (15mL),
3,4-methoxymethoxybenzaldehyde (2mmol), pyrrolidine (catalytic amount), and acetic acid
(catalytic amount) were added and the resulting solution was heated to reflux until absence of
3,4-methoxymethoxybenzaldehyde (checked by TLC). The solvent was evaporated under vacuum
and the mixture was diluted with H2O and extracted with ethyl acetate. The combined organic
fractions were washed with brine, dried with sodium sulfate, and concentrated in vacuo. Purification
of the crude product by column chromatography (PE/EA) afforded the title compound 8. Yield: 71%;
Yellow solid; mp 155–156 ºC; 1H NMR (400MHz, CDCl3) δ 8.16 (d, 2H, J = 8.7 Hz), 7.51 (d, 1H, J
= 15.4 Hz), 7.19-7.40 (m, 5H), 6.62 (d, 1H, J = 15.4 Hz), 5.27 (d, 4H, J = 13.2 Hz), 3.53 (d, 6H, J =
6.6 Hz), 3.23-3.39 (m, 4H); 13C NMR (100MHz, DMSO-d6) δ 149.0, 146.8, 145.3, 145.0, 133.6,
132.4, 130.1, 130.1, 124.2, 123.9, 123.9, 123.3, 111.3, 108.1, 96.1, 96.1, 54.1, 53.9, 51.0, 34.3; MS
(ESI) m/z 422.11 [M+H]+ 444.11 [M+Na]+.
4.1.4. Synthesis of (E)-3,4-diacetoxystyryl 4-aminophenethyl sulfone (9)
To a solution of (E)-3,4-diacetoxystyryl 4-nitrophenethyl sulfone (7, 0.5mmol) in
dichloromethane (20mL), Palladium/C (20mg) were added and the resulting solution was allowed to
stir under hydrogen gas for 3h at room temperature. The solvent was evaporated under vacuum and
the mixture was diluted with H2O and extracted with ethyl acetate. The combined organic fractions
were washed with brine, dried with sodium sulfate, and concentrated in vacuo. The crude product of
9 was washed with petroleum ether. Yield: 82%; Yellow oil; 1H NMR (400MHz, CDCl3) δ 7.46 (d,
1H, J = 15.4 Hz), 7.35 (d, 1H, J = 1.6 Hz), 7.22 (d, 1H, J = 15.4 Hz), 6.48-7.19 (m, 6H), 4.93 (s, 2H),
3.29 (m, 2H), 2.80 (m, 2H), 2.32 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 168.7, 168.7, 145.6,
143.7, 143.2, 133.7, 132.4, 129.3, 129.0, 129.0, 124.3, 124.1, 122.8, 115.3, 115.3, 55.7, 28.3, 20.6,
20.5; HRMS (ESI) m/z calcd for C20H21NNaO6S [M+Na]+: 426.0987, found: 426.0988.
4.1.5. Synthesis of (E)-4-(2-((3,4-bis(methoxymethoxy)styryl)sulfinyl)ethyl)aniline (10)
To a solution of (E)-1,2-bis(methoxymethoxy)-4-(2-((4-nitrophenethyl)sulfinyl)vinyl)benzene (8,
0.5mmol) in ethanol (with 16.7% water, 20mL), sodium hydrosulfite (8mmol) were added several
times and the resulting solution was heated to reflux until absence of compound 8 (checked by TLC).
The solvent was evaporated under vacuum and the mixture was diluted with H2O and extracted with
ethyl acetate. The combined organic fractions were washed with brine, dried with sodium sulfate,
and concentrated in vacuo. The crude product of 10 was washed with petroleum ether. Yield: 45%;

Yellow oil; 1H NMR (400MHz, CDCl3) δ 7.48 (d, 1H, J = 15.4 Hz), 6.63-7.29 (m, 7H), 6.57 (d, 1H,
J = 15.4 Hz), 5.27 (d, 4H, J = 4.4 Hz), 4.84 (s, 2H), 3.53 (s, 6H), 3.01-3.31 (m, 4H); 13C NMR
(100MHz, DMSO-d6) δ 149.1, 146.9, 145.3, 134.0, 129.8, 128.9, 128.9, 128.8, 122.2, 121.3, 115.4,
115.4, 111.5, 108.2, 96.5, 96.3, 54.1, 54.0, 51.2, 34.1; MS (ESI) m/z 392.16 [M+H]+.
4.1.6. General synthesis of (E)-3,4-diacetoxystyryl 4-acylaminophenethyl sulfones (11a – 11i)
To a solution of (E)-3,4-diacetoxystyryl 4-aminophenethyl sulfone (9, 0.1mmol) in
dichloromethane (10mL), trimethylamine (0.2mmol), and various acid chlorides (0.22mmol) were
added under the ice bath. The resulting solution was allowed to stir for 2h. The solvent was
evaporated under vacuum and the mixture was diluted with H2O and extracted with ethyl acetate.
The combined organic fractions were washed with brine, dried with sodium sulfate, and
concentrated in vacuo. Purification of the crude product by column chromatography (PE/EA)
afforded the target compounds 11a to 11i.
4.1.6.1. (E)-3,4-diacetoxystyryl 4-benzamidophenethyl sulfone (11a) 75%; Yellow solid; mp
92–93 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.21 (s, 1H), 6.79-7.95 (m, 14H), 3.42-3.46 (m, 2H),
2.94-2.98 (m, 2H), 2.33 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 168.6, 168.4, 165.2, 144.3, 142.7,
141.6, 138.2, 133.7, 133.3, 131.9, 131.7, 129.9, 129.9, 129.2, 129.2, 129.0, 129.0, 127.3, 124.7,
124.0, 120.8, 120.8, 114.9, 55.8, 28.1, 20.7, 20.6; MS (ESI) m/z 530.31 [M+Na]+; HRMS (ESI) m/z
calcd for C27H25NNaO7S [M+Na]+: 530.1249, found: 530.1251.
4.1.6.2. (E)-3,4-diacetoxystyryl 4-4-methoxylbenzamidophenethyl sulfone (11b) 49%; Buff
white solid; mp 106–107 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.04 (s, 1H), 7.06-7.95 (m, 13H),
3.84 (s, 3H), 3.49 (m, 2H), 2.98 (m, 2H), 2.30 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 168.6,
168.5, 165.3, 162.3, 144.4, 142.8, 141.7, 138.3, 133.4, 131.8, 130.0, 130.0, 129.1, 129.1, 128.0,
127.4, 124.8, 124.1, 120.9, 120.9, 115.0, 114.0, 114.0, 55.9, 55.3, 28.1, 20.8, 20.7; MS (ESI) m/z
560.21 [M+Na]+; HRMS (ESI) m/z calcd for C28H27NNaO8S [M+Na]+: 560.1355, found: 560.1356.
4.1.6.3. (E)-3,4-diacetoxystyryl 4-4-fluorobenzamidophenethyl sulfone (11c) 39%; Buff white
solid; mp 105–106 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.10 (s, 1H), 6.80-8.12 (m, 13H), 3.42 (m,
2H), 2.94 (m, 2H), 2.33 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 168.8, 168.6, 165.4, 144.7, 142.9,
141.8, 141.2, 138.2, 133.5, 131.9, 131.8, 130.1, 130.1, 129.8, 129.8, 129.2, 129.2, 127.2, 124.9,
124.2, 120.8, 120.8, 115.1, 56.0, 28.2, 21.0, 20.8; MS (ESI) m/z 548.30 [M+Na]+; HRMS (ESI) m/z
calcd for C27H24FNNaO7S [M+Na]+: 548.1155, found: 548.1152.
4.1.6.4. (E)-3,4-diacetoxystyryl 4-3.5-difluorobenzamidophenethyl sulfone (11d) 36%; Yellow
oil; 1H NMR (400MHz, DMSO-d6) δ 10.14 (s, 1H), 6.83-7.79 (m, 12H), 3.44 (m, 2H), 2.96 (m, 2H),
2.33 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 164.7, 161.0, 161.0, 146.5, 145.9, 137.4, 135.1,
135.0, 134.5, 129.2, 127.9, 127.9, 124.2, 123.2, 121.5, 121.5, 117.2, 115.2, 110.1, 110.1, 108.1, 60.7,

27.5, 20.9, 20.8; MS (ESI) m/z 566.08 [M+Na]+; HRMS (ESI) m/z calcd for C27H23F2NNaO7S
[M+Na]+: 566.1061, found: 566.1058.
4.1.6.5. (E)-3,4-diacetoxystyryl 4-cyclohexylamidophenethyl sulfone (11e) 47%; Buff white
solid; mp 98–99 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.12 (s, 1H), 6.74-7.85 (m, 9H), 3.44 (m,
2H), 2.95 (m, 2H), 2.29 (s, 6H), 1.20-1.76 (m, 11H); 13C NMR (100MHz, DMSO-d6) δ 174.6, 149.3,
146.1, 143.9, 138.4, 130.4, 130.0, 130.0, 124.3, 125.9, 125.9, 122.5, 119.6, 119.6, 116.2, 115.7, 55.5,
45.3, 29.6, 29.6, 28.2, 25.9, 25.8, 25.7; MS (ESI) m/z 536.22 [M+Na]+; HRMS (ESI) m/z calcd for
C27H31NNaO7S [M+Na]+: 536.1719, found: 536.1718.
4.1.6.6. (E)-3,4-diacetoxystyryl 4-1-adamantaneformylamidophenethyl sulfone (11f) 18%;
Yellow solid; mp 104–105 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.04 (s, 1H), 6.66-8.14 (m, 9H),
3.54 (m, 2H), 3.12 (m, 2H), 2.14 (s, 6H), 1.60-2.00 (m, 15H); 13C NMR (100MHz, DMSO-d6) δ
174.6, 169.3, 169.3, 149.3, 146.1, 143.9, 148.4, 132.9, 129.2, 129.1, 129.1, 124.3, 122.5, 122.4,
119.6, 119.6, 119.5, 116.2, 115.8, 55.6, 45.3, 29.6, 29.6, 29.6, 28.2, 25.9, 25.7, 25.7, 25.7, 24.7, 24.7,
24.7; MS (ESI) m/z 566.24 [M+H]+ 588.17 [M+Na]+; HRMS (ESI) m/z calcd for C31H35NNaO7S
[M+Na]+: 588.2032, found: 588.2039.
4.1.6.7. (E)-3,4-diacetoxystyryl 4-4-methylbenzamidophenethyl sulfone (11g) 25%; White solid;
mp 108–109 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.12 (s, 1H), 6.78-7.96 (m, 13H), 3.44 (m, 2H),
2.95 (m, 2H), 2.43 (s, 3H), 2.34 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 168.8, 168.6, 165.4,
144.5, 142.9, 141.8, 141.2, 138.4, 133.5, 131.9, 131.8, 130.1, 130.1, 129.4, 129.4, 129.2, 129.2,
127.5, 124.9, 124.2, 121.0, 121.0, 115.1, 56.0, 28.3, 23.5, 20.9, 20.8; MS (ESI) m/z 544.11 [M+Na]+;
HRMS (ESI) m/z calcd for C28H27NNaO7S [M+Na]+: 544.1406, found: 544.1399.
4.1.6.8. (E)-3,4-diacetoxystyryl 4-4-nitrobenzamidophenethyl sulfone (11h) 35%; Yellow solid;
mp 126–127 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.21 (s, 1H), 6.80-7.95 (m, 13H), 3.43-3.46 (m,
2H), 2.94-2.98 (m, 2H), 2.33 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 168.6, 168.4, 165.7, 144.0,
142.7, 141.6, 138.2, 133.7, 133.3, 131.9, 131.6, 129.9, 129.9, 129.3, 129.3, 129.0, 129.0, 127.3,
124.7, 124.0, 120.8, 120.8, 114.9, 55.8, 28.1, 20.7, 20.6; MS (ESI) m/z 575.29 [M+Na]+; HRMS
(ESI) m/z calcd for C27H24N2NaO9S [M+Na]+: 575.1100, found: 575.1101.
4.1.6.9. (E)-3,4-diacetoxystyryl 4-2-thiopheneacetylamidophenethyl sulfone (11i) 16%; Yellow
brown solid; mp 92–93 ºC; 1H NMR (400MHz, DMSO-d6) δ 7.00-8.03 (m, 12H), 6.80 (s, 1H), 3.48
(s, 2H), 3.43 (m, 2H), 2.95 (m, 2H), 2.30 (s, 6H); 13C NMR (100MHz, DMSO-d6) δ 169.5, 168.5,
168.5, 149.3, 146.1, 143.9, 140.2, 138.4, 135.9, 133.3, 130.1, 129.2, 129.2, 129.0, 128.4, 127.3,
124.7, 124.2, 120.8, 120.8, 113.5, 55.4, 45.5, 28.0, 20.7, 20.5; MS (ESI) m/z 550.18 [M+Na]+;
HRMS (ESI) m/z calcd for C26H25NNaO7S2 [M+Na]+: 550.0970, found: 550.0968.
4.1.7. General synthesis of (E)-3,4-dihydroxystyryl 4-acylaminophenethyl sulfones (13a – 13i)

To a solution of (E)-3,4-diacetoxystyryl 4-acylaminophenethyl sulfones (11a – 11i, 0.06mmol) in
methanol (10mL), potassium carbonate (0.18mmol) was added and the resulting solution was
allowed to stir for 3h at room temperature. The solvent was evaporated under vacuum and the
mixture was diluted with H2O and extracted with ethyl acetate. The combined organic fractions
were washed with brine, dried with sodium sulfate, and concentrated in vacuo. Purification of the
crude product by column chromatography (PE/EA) afforded the target compounds 13a to 13i.
4.1.7.1. (E)-3,4-dihydroxystyryl 4-benzamidophenethyl sulfone (13a) 65%; White solid; mp
104–105 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.12 (s, 1H), 9.72 (s, 1H), 9.21 (s, 1H), 6.80-7.94 (m,
14H), 3.44 (m, 2H), 2.96 (m, 2H); 13C NMR (100MHz, DMSO-d6) δ 165.9, 149.4, 146.1, 144.0,
138.1, 135.4, 133.8, 132.0, 129.1, 129.1, 128.8, 128.8, 128.1, 128.1, 124.4, 122.5, 122.5, 120.9,
120.9, 116.2, 115.8, 55.5, 28.2; MS (ESI) m/z 422.25 [M-H]-; HRMS (ESI) m/z calcd for
C23H20NO5S [M-H]-: 422.1062, found: 422.1062.
4.1.7.2. (E)-3,4-dihydroxystyryl 4-4-methoxylbenzamidophenethyl sulfone (13b) 58%; Buff
white solid; mp 108–109 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.20 (s, 1H), 9.71 (s, 1H), 9.22 (s,
1H), 6.81-7.96 (m, 13H), 3.81 (s, 3H), 3.43 (m, 2H), 2.95 (m, 2H); 13C NMR (100MHz, DMSO-d6)
δ 165.3, 162.3, 149.3, 146.1, 144.0, 138.3, 133.6, 130.4, 130.0, 120.0, 129.1, 129.1, 127.4, 123.9,
122.5, 120.9, 120.9, 116.2, 115.7, 114.1, 114.1, 55.9, 55.5, 28.2; MS (ESI) m/z 452.10 [M-H]-;
HRMS (ESI) m/z calcd for C24H22NO6S [M+Na]+: 452.1168, found: 452.1166.
4.1.7.3. (E)-3,4-dihydroxystyryl 4-4-fluorobenzamidophenethyl sulfone (13c) 61%; Buff white
oil; 1H NMR (400MHz, DMSO-d6) δ 10.21 (s, 1H), 9.71 (s, 1H), 9.20 (s, 1H), 6.79-8.12 (m, 13H),
3.43 (m, 2H), 2.97 (m, 2H); 13C NMR (100MHz, DMSO-d6) δ 164.7, 149.5, 146.5, 144.5, 138.0,
135.5, 134.2, 132.1, 129.2, 129.2, 128.8, 128.8, 124.8, 123.2, 121.5, 121.5, 115.5, 115.2, 55.7, 28.5;
MS (ESI) m/z 440.26 [M-H]-; HRMS (ESI) m/z calcd for C23H19FNO5S [M-H]-: 440.0968, found:
440.0972.
4.1.7.4. (E)-3,4-dihydroxystyryl 4-3.5-difluorobenzamidophenethyl sulfone (13d) 56%; Yellow
oil; 1H NMR (400MHz, DMSO-d6) δ 10.14 (s, 1H), 9.71 (s, 1H), 9.22 (s, 1H), 6.83-7.79 (m, 12H),
3.44 (m, 2H), 2.96 (m, 2H); 13C NMR (100MHz, DMSO-d6) δ 164.7, 161.0, 161.0, 146.5, 145.9,
137.4, 135.1, 135.0, 134.5, 129.2, 127.9, 127.9, 124.2, 123.2, 121.5, 121.5, 117.2, 115.2, 110.1,
110.1, 108.1, 60.7, 27.5; MS (ESI) m/z 458.21 [M-H]-; HRMS (ESI) m/z calcd for C23H18F2NO5S
[M-H]-: 458.0874, found: 458.0878.
4.1.7.5. (E)-3,4-dihydroxystyryl 4-cyclohexylamidophenethyl sulfone (13e) 65%; White solid;
mp 83–84 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.22 (s, 1H), 9.72 (s, 1H), 9.22 (s, 1H), 6.80-7.80
(m, 9H), 3.56 (m, 2H), 2.92 (m, 2H), 1.19-2.32 (m, 11H); 13C NMR (100MHz, DMSO-d6) δ 174.7,
149.3, 146.1, 143.9, 138.4, 130.4, 129.1, 129.1, 124.3, 123.9, 122.4, 119.6, 119.4, 116.2, 115.7, 55.5,

45.3, 29.6, 29.6, 28.2, 25.8, 25.7, 25.7; MS (ESI) m/z 428.32 [M-H]-; HRMS (ESI) m/z calcd for
C23H26NO5S [M-H]-: 428.1532, found: 428.1532.
4.1.7.6. (E)-3,4-dihydroxystyryl 4-1-adamantaneformylamidophenethyl sulfone (13f) 59%;
Yellow solid; mp 122–123 ºC; 1H NMR (400MHz, DMSO-d6) δ 9.65 (s, 1H), 9.05 (s, 1H), 9.05 (s,
1H), 6.76-8.13 (m, 9H), 3.54 (m, 2H), 3.12 (m, 2H), 1.55-2.07 (m, 15H); 13C NMR (100MHz,
DMSO-d6) δ 174.6, 149.3, 146.0, 143.5, 138.4, 132.9, 129.2, 129.1, 124.3, 121.5, 121.4, 119.6,
119.6, 119.5, 116.0, 115.8, 55.6, 45.3, 29.6, 29.6, 29.6, 28.1, 25.9, 25.9, 25.9, 25.7, 25.7, 25.7; MS
(ESI) m/z 480.17 [M-H]-; HRMS (ESI) m/z calcd for C27H30NO5S [M-H]-: 480.1845, found:
480.1843.
4.1.7.7. (E)-3,4-dihydroxystyryl 4-4-methylbenzamidophenethyl sulfone (13g) 58%; Light
1
brown solid; mp 119–120 ºC; H NMR (400MHz, DMSO-d6) δ 10.21 (s, 1H), 9.51 (s, 2H),
6.79-7.86 (m, 13H), 3.44 (m, 2H), 2.95 (m, 2H), 2.38 (s, 3H); 13C NMR (100MHz, DMSO-d6) δ
165.7, 149.4, 146.1, 144.0, 142.0, 138.2, 133.7, 132.5, 129.4, 129.4, 129.4, 129.1, 129.1, 128.1,
128.1, 124.3, 122.5, 120.9, 120.9, 116.2, 115.8, 55.5, 28.2, 21.4; MS (ESI) m/z 436.15 [M-H]-;
HRMS (ESI) m/z calcd for C24H22NO5S [M-H]-: 436.1219, found: 436.1219.
4.1.7.8. (E)-3,4-dihydroxystyryl 4-4-nitrobenzamidophenethyl sulfone (13h) 60%; White solid;
1
mp 138–139 ºC; H NMR (400MHz, DMSO-d6) δ 10.21 (s, 1H), 9.72 (s, 1H), 9.21 (s, 1H),
6.78-8.39 (m, 13H), 3.44 (m, 2H), 2.95 (m, 2H); 13C NMR (100MHz, DMSO-d6) δ 164.7, 151.3,
146.5, 145.9, 136.8, 135.1, 135.0, 134.5, 129.6, 129.6, 127.9, 127.9, 124.2, 124.0, 123.2, 124.0,
121.5, 121.5, 117.2, 115.2, 60.7, 27.5; MS (ESI) m/z 467.08 [M-H]-; HRMS (ESI) m/z calcd for
C23H19N2O7S [M-H]-: 467.0913, found: 467.0911.
4.1.7.9. (E)-3,4-dihydroxystyryl 4-2-thiopheneacetylamidophenethyl sulfone (13i) 59%; Yellow
brown solid; mp 87–88 ºC; 1H NMR (400MHz, DMSO-d6) δ 9.73 (s, 1H), 9.24 (s, 1H), 6.81-7.79
(m, 12H), 5.31 (s, 1H), 3.54 (m, 4H), 2.96 (m, 2H); 13C NMR (100MHz, DMSO-d6) δ 158.8, 146.5,
145.9, 137.4, 135.1, 135.0, 134.5, 129.2, 127.9, 127.9, 124.2, 123.2, 121.5, 121.5, 117.2, 115.2,
110.1, 110.1, 108.1, 60.7, 38.0, 27.6; MS (ESI) m/z 442.07 [M-H]-; HRMS (ESI) m/z calcd for
C22H20NO5S2 [M-H]-: 442.0783, found: 442.0778.
4.1.8. General synthesis of (E)-N-(4-(2-((3,4-bis(methoxymethoxy)styryl)sulfinyl)ethyl)phenyl)
amides (12a – 12f)
To a solution of (E)-4-(2-((3,4-bis(methoxymethoxy)styryl)sulfinyl)ethyl)aniline (10, 0.1mmol)
in dichloromethane (10mL), trimethylamine (0.2mmol), and various acid chlorides (0.22mmol)
were added under the ice bath. The resulting solution was allowed to stir for 2h. The solvent was
evaporated under vacuum and the mixture was diluted with H2O and extracted with ethyl acetate.
The combined organic fractions were washed with brine, dried with sodium sulfate, and

concentrated in vacuo. The crude products of 12a to 12f were washed with petroleum ether and were
immediately added to the next step.
4.1.9. General synthesis of (E)-3,4-dihydroxystyryl 4-acylaminophenethyl sulfoxides (14a –
14f)
To a solution of (E)-N-(4-(2-((3,4-bis(methoxymethoxy)styryl)sulfinyl)ethyl)phenyl)amides (12a
– 12f, 0.06mmol) in methanol (10mL), hydrochloric acid (catalytic amount) was added and the
resulting solution was heated to reflux for 3h. The solvent was evaporated under vacuum and the
mixture was diluted with H2O and extracted with ethyl acetate. The combined organic fractions
were washed with brine, dried with sodium sulfate, and concentrated in vacuo. Purification of the
crude product by column chromatography (PE/EA) afforded the target compounds 14a to 14f.
4.1.9.1. (E)-3,4-dihydroxystyryl 4-benzamidophenethyl sulfoxide (14a) 15%; White solid; mp
97–98 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.21 (s, 1H), 9.72 (s, 1H), 9.21 (s, 1H), 6.79-7.95 (m,
14H), 2.74-3.04 (m, 4H); 13C NMR (100MHz, DMSO-d6) δ 166.0, 149.4, 146.1, 143.9, 138.1, 135.5,
133.9, 132.0, 129.1, 129.1, 128.8, 128.8, 128.1, 128.1, 124.4, 122.5, 122.5, 120.9, 120.9, 116.2,
115.7, 50.3, 36.9; MS (ESI) m/z 406.10 [M-H]-; HRMS (ESI) m/z calcd for C23H20NO4S [M-H]-:
406.1113, found: 406.1112.
4.1.9.2. (E)-3,4-dihydroxystyryl 4-4-methoxylbenzamidophenethyl sulfoxide (14b) 11%;
Yellow oil; 1H NMR (400MHz, DMSO-d6) δ 10.27 (s, 1H), 9.71 (s, 1H), 9.21 (s, 1H), 6.81-8.44 (m,
13H), 3.81 (s, 3H), 2.74-3.04 (m, 4H); 13C NMR (100MHz, DMSO-d6) δ 165.5, 162.5, 149.5, 146.3,
144.1, 138.5, 133.8, 130.6, 130.2, 130.2, 129.3, 129.3, 127.6, 124.1, 122.6, 121.1, 121.1, 116.4,
115.9, 114.3, 114.3, 56.1, 51.6, 33.1; MS (ESI) m/z 436.11 [M-H]-; HRMS (ESI) m/z calcd for
C24H22NO5S [M+Na]+: 436.1219, found: 436.1216.
4.1.9.3. (E)-3,4-dihydroxystyryl 4-4-fluorobenzamidophenethyl sulfoxide (14c) 17%; Yellow
oil; 1H NMR (400MHz, DMSO-d6) δ 10.27 (s, 1H), 9.72 (s, 1H), 9.21 (s, 1H), 6.79-8.44 (m, 13H),
2.76-3.00 (m, 4H); 13C NMR (100MHz, DMSO-d6) δ 168.2, 165.9, 149.5, 146.3, 144.2, 142.1,
138.4, 133.9, 132.7, 129.5, 129.5, 129.2, 128.3, 128.3, 124.5, 122.6, 122.6, 121.1, 121.1, 126.4,
115.9, 52.8, 31.9; MS (ESI) m/z 424.09 [M-H]-; HRMS (ESI) m/z calcd for C23H19FNO4S [M-H]-:
424.1019, found: 424.1022.
4.1.9.4. (E)-3,4-dihydroxystyryl 4-3.5-difluorobenzamidophenethyl sulfoxide (14d) 23%;
Yellow oil; 1H NMR (400MHz, DMSO-d6) δ 10.27 (s, 1H), 9.72 (s, 1H), 9.22 (s, 1H), 6.80-8.43 (m,
12H), 2.76-3.04 (m, 4H); 13C NMR (100MHz, DMSO-d6) δ 165.9, 161.0, 161.0, 149.4, 146.1, 144.0,
138.1, 135.5, 133.9, 132.0, 129.1, 128.9, 128.9, 122.5, 122.5, 121.0, 116.2, 115.7, 115.2, 110.6,
108.7, 108.7, 50.4, 36.9; MS (ESI) m/z 442.08 [M-H]-; HRMS (ESI) m/z calcd for C23H18F2NO4S
[M-H]-: 442.0925, found: 442.0928.

4.1.9.5. (E)-3,4-dihydroxystyryl 4-cyclohexylamidophenethyl sulfoxide (14e) 10%; Yellow oil;
1H NMR (400MHz, DMSO-d6) δ 10.27 (s, 1H), 9.72 (s, 1H), 9.22 (s, 1H), 6.80-7.94 (m, 9H),
2.74-3.04 (m, 4H), 1.19-1.76 (m, 11H); 13C NMR (100MHz, DMSO-d6) δ 174.8, 149.5, 146.3,
144.1, 138.6, 133.1, 129.4, 129.3, 124.5, 122.7, 122.6, 119.8, 119.7, 116.4, 116.0, 52.3, 45.5, 34.1,
29.8, 29.8, 26.1, 25.9, 25.9; MS (ESI) m/z 412.15 [M-H]-; HRMS (ESI) m/z calcd for C23H26NO4S
[M-H]-: 412.1583, found: 412.1582.
4.1.9.6. (E)-3,4-dihydroxystyryl 4-1-adamantaneformylamidophenethyl sulfoxide (14f) 15%;
Yellow solid; mp 111–112 ºC; 1H NMR (400MHz, DMSO-d6) δ 10.05 (s, 1H), 9.65 (s, 1H), 9.05 (s,
1H), 6.79-7.96 (m, 9H), 2.74-3.04 (m, 4H), 1.60-2.00 (m, 15H); 13C NMR (100MHz, DMSO-d6) δ
174.7, 149.3, 146.1, 143.9, 138.4, 130.4, 129.1, 129.1, 124.3, 123.9, 122.5, 122.5, 119.6, 119.6,
116.2, 115.7, 52.4, 35.7, 35.4, 35.4, 34.3, 34.3, 32.9, 32.5, 32.1, 32.1, 25.9; MS (ESI) m/z 464.18
[M-H]-; HRMS (ESI) m/z calcd for C27H30NO4S [M-H]-: 464.1896, found: 464.1893.
4.2. Biology
4.2.1. Cytotoxicity assays
The cytotoxicity of all target compounds were assessed via measurement of the amount of
TZM-bl cells after exposure to the compounds for 2 to 3 days. In short, the target compounds were
dissolved in DMSO cell freezing medium. TZM-bl cells were seeded in 96-well culture plates at 1 ×
105 cells per mL (100μL per well). The cells were treated with various concentrations (1000μM,
200μM, 40μM, 8μM, and 1.6μM) of each compounds and were cultivated under 5% CO2 at 37°C.
Three wells were used in parallel for each concentration. Cells treated with culture medium were
used as a negative control. Two days later, the culture medium was discarded and 10μL of coloring
agent CCK-8 solution was added to each well before incubation was allowed to last for another
three to four hours. The absorbance (A, at 450nm) was measured by a microplate reader. The (%)
survival of TZM-bl cells was calculated using the following formula: (A450 of the experimental
group/ A450 of the negative control group) × 100%.
4.2.2. Determination of antivirus activity against R5 HIV-1 strains
TZM-bl cells, the same type used in cytotoxicity assays, were seeded in 96-well plates. Complete
medium were added to all sample wells, viral control wells and cell control wells. Twelve hours later,
the culture medium was discarded before the solutions of target compounds were diluted to 50μM
and added to sample wells. Two wells were used in parallel for each compound. HIV-1 Bal (R5)
virus (200TCID50) were added into all sample wells and virus control wells and DEAE was added
to a final concentration of 10μg/mL. The infected cells were cultivated for 3 days under 5% CO2 at
37°C. The luciferase assay substrate were dissolved by luciferase lysis buffer and were added
(100μL per well) into all experiment wells which had 100μL of the culture medium discarded
beforehand. The luciferase assay system was shielded from light and was allowed to react for 2
minutes at room temperature. The mixtures in all wells were blown to well-distributed and

transferred to a black test plate for 150μL for each well. The results were measured by a
fluorescence detector and the % inhibition of virus’ infection was represented with the decrease in
fluorescent intensity.
4.2.3. SPR binding assays
The SPR binding assays were performed as previously described by using SPR biosensor
technology. The experiments were carried out on a Biacore T200 instrument that contains dual flow
cells. Immobilization of IN proteins to sensor chip CM5 (Biacore) was carried out by primary amine
coupling method resulting in an immobilization level of approximately 10000RU. By using
automatic corrections of Biacore T200 Evaluation Software, all the raw sensorgrams were
processed along with solvent corrected. The specific binding figures of compounds to immobilized
IN were obtained after subtracting response signal from control flow cell. All Biacore data were
collected at 25°C with PBST as running buffer at a constant flow of 30μL/min. The equilibrium
dissociation constants (KD) evaluating the binding affinity between IN and compounds were
determined using 1:1 binding model and the curve fitting efficiency was checked by χ2 and residual
plots.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (Grants 20972011,
21042009, and 21172014) and Ministry of Science and Technology of China (Grant
2009ZX09301-010).
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Binding with IN.
Binding with CCR5.